To evaluate the optimal substitution ratio of organic fertilizer for chemical nitrogen fertilizer and its underlying mechanisms, a pot experiment was conducted in the rhizosphere soil of oat (Avena sativa) on the Qinghai–Tibet Plateau. Five treatments were established: CK (control), T1 (chemical fertilizer alone), T2 (100% organic fertilizer substitution for chemical nitrogen fertilizer), T3 (30% organic fertilizer substitution for chemical nitrogen fertilizer), and T4 (60% organic fertilizer substitution for chemical nitrogen fertilizer). We analyzed soil carbon fractions, microbial community structure, carbon-cycling enzyme activities, and yield responses and applied partial least squares–structural equation modeling (PLS-SEM) to identify key regulatory pathways. The results showed that 30% organic substitution (T3) was associated with optimized soil carbon pools, improved microbial community composition, and enhanced carbon-cycling enzyme activities, while reducing the abundance of potentially harmful fungi. Structural equation modeling indicated that β-glucosidase activity and the relative abundance of Proteobacteria were the primary drivers of yield, together explaining 76% of its variation. The ecosystem multifunctionality index (EMF) was significantly and positively correlated with yield. In summary, under the conditions of this experiment, 30% organic fertilizer substitution achieved a favorable balance between soil ecological functions and crop yield, providing a valuable reference for sustainable nutrient management in oat production in high-altitude cold regions.